Genomic instability of ovarian cancer (OvCa) cells and the resulting frequent loss or reduction of HLA expression and function facilitate immune avoidance of this aggressive cancer. The goal of this Project is to develop an effective mode of immunotherapy capable of targeting both MHC-positive and MHC-negative variants of OvCa and counteracting local immune suppression, known to contribute to poor prognosis in OvCa patients. We observed that type-1-polarized DCs (?DC1s), a novel type of DCs developed by our group, effectively cross-present OvCa-related antigens and induce high numbers of MHC class l-restricted CTLs capable of recognizing defined OvCa-related antigenic epitopes. However ?DC1-induced CTLs also express elevated levels of NK receptors, NKG2D- and DNAM1. Such NK cell-like (n)CTLs recognize OvCa cells in both MHC class l-restricted- and MHC class l-unrestricted fashion. IFN? produced by ?DC1-activated nCTLs has a strong positive impact on OvCa microenvironment, suppressing the chemokines that attract undesirable Treg cells and MDSCs and enhancing the production of the chemokines that attract additional effector cells. Based on these preliminary data, we hypothesize that autologous tumor-loaded ?DCIs used both as vaccines and as ex-vivo inducers of OvCa-specific CTLs for adoptive T cell transfer in OvCa can be useful in the immunotherapy of patients treated with primary chemotherapy (which may sensitize tumor cells for the NKG2D- and DNAM-1-mediated recognition). We will test the hypothesis that local adoptive transfer of ex-vivo-sensitized (n)CTLs will amplify the effects of systemic vaccination against OvCa, by promoting direct elimination of residual tumor cells and the attraction of additional (vaccination induced) T cells. In Specific Aim 1, we will evaluate the mechanisms involved in the induction of OvCa-specific NKG2D{high}/DNAM-1{high} (n)CTLs by ?DCIs and the mechanisms of their TCR-dependent and TCR-independent recognition and killing of OvCa cells. In Specific Aim 2, we will evaluate the regulation of the NK cell-type receptors ligands (MICA/B, ULBP1-6, PVR/CD155 and Necfin-2/CD112) on OvCa cells by chemotherapy, CTL/NK-produced cytokines and additional clinically-applicable biologic agents, and will evaluate the potential for additional sensitization of OvCa cells for (n)CTL-mediated killing. In Specific Aim 3, we will evaluate in phase I clinical trial (24 high-risk patients with advanced primary OvCa) the feasibility and safety of combined immunotherapy of advanced OvCa involving vaccination with autologous tumor-loaded ?DCIs and intraperitoneal adoptive transfer of increasing numbers ex-vivo-induced OvCa-specific (n)CTLs, in the context of systemic and intraperitoneal chemotherapy.